Raymond Teck Ho Lee

Bacterial Peptidoglycan Triggers Candida albicans Hyphal Growth by Directly Activating the Adenylyl Cyclase Cyr1p

Human serum potently induces hyphal development of the polymorphic fungal pathogen Candida albicans, a phenotype that contributes critically to infections. The fungal adenylyl cyclase Cyr1p is a key component of the cAMP/PKA-signaling pathway that controls diverse infection-related traits, including hyphal morphogenesis. However, identity of the serum hyphal inducer(s) and its fungal sensor remain unknown. Our initial analyses of active serum fractions revealed signs of bacterial peptidoglycan (PGN)-like molecules. Here, we show that several purified and synthetic muramyl dipeptides (MDPs), subunits of PGN, can strongly promote C. albicans hyphal growth. Analogous to PGN recognition by the mammalian sensors Nod1 and Nod2 through their leucine-rich-repeat (LRR) domain, we show that MDPs activate Cyr1p by directly binding to its LRR domain. Given the abundance of PGN in the intestine, a natural habitat and invasion site for C. albicans, our findings have important implications for the mechanisms of infection by this pathogen.

Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin‐dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase‐activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1‐dependent hyperphosphorylation. Using the analog‐sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Δ mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28‐dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.

Candida albicans hyphal morphogenesis occurs in Sec3p-independent and Sec3p-dependent phases separated by septin ring formation

The growing tips of Candida albicans hyphae are sites of polarized exocytosis. Mammalian septins have been implicated in regulating exocytosis and C. albicans septins are known to localize at hyphal tips, although their function here is unknown. Here, we report that C. albicans cells deleted of the exocyst subunit gene SEC3 can grow normal germ tubes, but are unable to maintain tip growth after assembly of the first septin ring, resulting in isotropic expansion of the tip. Deleting either of the septin genes CDC10 or CDC11 caused Sec3p mislocalization and surprisingly, also restored hyphal development in the sec3Δ mutant without rescuing the temperature sensitivity. Co-immunoprecipitation experiments detected association of the septin Cdc3p with the exocyst subunits Sec3p and Sec5p. Our results reveal that C. albicans hyphal development occurs through Sec3p-independent and dependent phases, and provide strong genetic and biochemical evidence for a role of septins in polarized exocytosis.

Dermal fin rays and scales derive from mesoderm, not neural crest

Summary Neural crest cells disperse throughout the embryonic head to generate diverse cell types of two classes: non-ectomesenchymal (including melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue cell fates). In contrast to cranial neural crest, trunk neural crest of amniotes generates only non-ectomesenchymal cell types. Anamniote trunk neural crest, however, has been assumed to generate derivatives of both classes, including osteoblasts of dermal skeletal elements, which includes scales and fin rays. Through genetic lineage tracing in zebrafish, we present the first test of this assumption and find that trunk neural crest does not generate fin osteoblasts; rather, these derive from a late emerging population of paraxial mesoderm. Similarly we show that the mineralising cells of the scales are mesodermally derived, with no contribution from neural crest. Our data suggest that trunk/tail exoskeletal structures evolved through deployment of mesodermally derived mesenchyme, rather than neural crest.

An exclusively mesodermal origin of fin mesenchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme.

The neural crest is a multipotent stem cell population that arises from the dorsal aspect of the neural tube and generates both non-ectomesenchymal (melanocytes, peripheral neurons and glia) and ectomesenchymal (skeletogenic, odontogenic, cartilaginous and connective tissue) derivatives. In amniotes, only cranial neural crest generates both classes, with trunk neural crest restricted to non-ectomesenchyme. By contrast, it has been suggested that anamniotes might generate derivatives of both classes at all axial levels, with trunk neural crest generating fin osteoblasts, scale mineral-forming cells and connective tissue cells; however, this has not been fully tested. The cause and evolutionary significance of this cranial/trunk dichotomy, and its absence in anamniotes, are debated. Recent experiments have disputed the contribution of fish trunk neural crest to fin osteoblasts and scale mineral-forming cells. This prompted us to test the contribution of anamniote trunk neural crest to fin connective tissue cells. Using genetics-based lineage tracing in zebrafish, we find that these fin mesenchyme cells derive entirely from the mesoderm and that neural crest makes no contribution. Furthermore, contrary to previous suggestions, larval fin mesenchyme cells do not generate the skeletogenic cells of the adult fin, but persist to form fibroblasts associated with adult fin rays. Our data demonstrate that zebrafish trunk neural crest does not generate ectomesenchymal derivatives and challenge long-held ideas about trunk neural crest fate. These findings have important implications for the ontogeny and evolution of the neural crest.

MNN5 Encodes an Iron-Regulated α-1,2-Mannosyltransferase Important for Protein Glycosylation, Cell Wall Integrity, Morphogenesis, and Virulence in Candida albicans

ABSTRACT The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans α-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both α-1,2- and α-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Δ mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Δ mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Δ mutants resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.

Cell delamination in the mesencephalic neural fold and its implication for the origin of ectomesenchyme

The neural crest is a transient structure unique to vertebrate embryos that gives rise to multiple lineages along the rostrocaudal axis. In cranial regions, neural crest cells are thought to differentiate into chondrocytes, osteocytes, pericytes and stromal cells, which are collectively termed ectomesenchyme derivatives, as well as pigment and neuronal derivatives. There is still no consensus as to whether the neural crest can be classified as a homogenous multipotent population of cells. This unresolved controversy has important implications for the formation of ectomesenchyme and for confirmation of whether the neural fold is compartmentalized into distinct domains, each with a different repertoire of derivatives. Here we report in mouse and chicken that cells in the neural fold delaminate over an extended period from different regions of the cranial neural fold to give rise to cells with distinct fates. Importantly, cells that give rise to ectomesenchyme undergo epithelial-mesenchymal transition from a lateral neural fold domain that does not express definitive neural markers, such as Sox1 and N-cadherin. Additionally, the inference that cells originating from the cranial neural ectoderm have a common origin and cell fate with trunk neural crest cells prompted us to revisit the issue of what defines the neural crest and the origin of the ectomesenchyme.

Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.

Basal Keratinocytes Contribute to All Strata of the Adult Zebrafish Epidermis

The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened

The G‐protein‐coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock‐down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP‐dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase‐dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C‐terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho‐null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.